Human cardiac myosin regulatory light chain (RLC) mutations have been linked to a rare ventricular hypertrophy and skeletal muscle abnormality in familial hypertrophic cardiomyopathy (HCM). In order to understand the functional effects of these mutations, we have expressed the RLC mutations, Ala13Thr, Glu22Lys, Pro94Arg, along with artificial mutations on the phosphorylatable serine (Ser15Ala, Ser15Asp) in a bacteria expression system. The expressed RLCs were then exchanged into normal freshly purified human cardiac myosin with the native RLC depleted. The reconstituted myosins carrying different mutations on RLC were then tested in an in vitro motility assay. Purified human cardiac myosin has also been digested with papain to obtain subfragment 1 for ATPase assay. The preliminary data indicate that the light chain exchange procedures did not impair the myosin function as assessed by in vitro motility rate. Both Glu22Lys and Pro94Arg mutations resulted in slower motility rates whereas that of Ala13Thr was not altered. The ATPase activity for Glu22Lys seemed to be elevated significantly while other mutants did not change the ATPase activities. However, we were unable to prepare sufficient quantities of subfragment-one (S-1) by papain digestion of human cardiac myosin to carry out the desired kinetic experiments and the S-1 that was prepared was heterogeneous upon analysis by SDS gel electrophoresis. Therefore, we turned to the highly homologous porcine cardiac myosin as a starting procedure. Large quantities of myosin can be purified from slaughter house obtained material and a homogeneous preparation of S-1 was prepared. This material will be used to for light chain exchange to produce material for transient kinetic analysis of the consequences of HCM-causing mutations in both the essential and regulatory light chains.